Apoptotic cell death is a fundamentally important biological process that is required to maintain the integrity and homeostasis of multicellular organisms. Inappropriate and excessive apoptosis, however, underlies the etiology of many of the most intractable of human diseases. The apoptotic pathway is predominantly executed by a series of cysteine proteases designated the “caspases” (cysteinyl aspartate-specific proteinases). Caspases are intracellular protease enzymes that play significant roles in both cytokine maturation and programmed cell death (apoptosis) (see, Thornberry et al., Nature 1992, 356, 768-774; Thornberry et al. Chem. Biol. 1998, 5, R97-103). Specifically, caspases are responsible for the proteolytic degradation of more than 100 different protein substrates, including proteins involved in DNA repair, nuclear membrane integrity, and cell structural integrity.
The first caspase to be discovered was Interleukin-1β Converting Enzyme (ICE), now also known as caspase-1 (Thornberry et al. 1992, Nature 356:768-774; Cerretti et al. 1992, Science 256:97-99). Caspase-1 was initially identified as the protease that cleaves the immature pro-IL-1β polypeptide to produce the mature IL-1β polypeptide, a critical step that precedes secretion of IL-1β from the cell. Since IL-1β is an important mediator of inflammation, it has been suggested that disruption of caspase-1 activity may reduce the inflammatory response after exposure to an appropriate stimulant. This was shown to be the case in mice containing a “knockout” of the caspase-1 gene. These mice undergo normal development but are deficient in mounting a normal inflammatory response (Kuida et al. 1995, Science 267:2000-2003; Li et al. 1995, Cell 80:401-411). Even though the predominant role of caspase-1 appears to involve the inflammatory pathway, evidence indicates that it is also important for the apoptotic pathway, since these mice also show reduced levels of apoptosis when treated with chemicals that typically induce apoptosis.
To date, approximately eleven caspases have been identified in humans. Caspases have been broadly categorized into three main functional categories. Group I caspases (e.g. caspase-1, -4 and -5) are predominantly involved in the inflammatory response pathway, Group II caspases (e.g. caspase-3, -6, and -7) are the effector caspases, and Group III caspases (e.g. caspase-8, -9 and -2) are the initiator caspases (reviewed in Thomberry 1998, Current Biology 5:R97-103). The initiator caspases are typically located higher in the activation pathway, with one of their main functions being the activation of the effector caspases through cleavage at conserved Asp residues located immediately upstream of both the large and small subunits. Following cleavage, the large and small subunits rearrange to form a heterotetramer, which is the catalytically active form of the enzyme. Once activated, the caspases then proceed not only to proteolytically degrade a wide range of cellular proteins, but also to amplify the apoptotic response through a positive feedback mechanism whereby downstream caspases can cleave certain members of upstream caspases.
The crystal structures of the mature active forms of caspase-1 (Wilson et al. 1994, Nature 370:270-275), caspase-3 (Rotunda et al. 1996, Nature Struct. Biol. 3:619-625; Lee et. al. 2001, J. Med. Chem. 44:2015-2126), caspase-7 (Wei et. al. 2000, Chem. Biol. 7:423-432), and caspase-8 (Watt et al. 1999, Structure 7:1135-1143; Blanchard et al. 1999, Structure 7:1125-1133) have been solved, in each case in the presence of a bound peptide or small molecule inhibitor. These structures have helped researchers understand the mechanism of peptide hydrolysis and have also aided in the design of small molecule competitive inhibitors. For example, the minimal substrate for each caspase was determined to consist of a tetrapeptide sequence (Thornberry et. al. 1997, J. Biol. Chem. 272:17907-17911). In addition, while the Asp residue at position P1 is conserved across all three caspases, amino acid variations are found in the P2-P4 positions. For example, caspase-3 prefers a negatively charged amino acid in the P4 position (Asp), compared to caspase-1 which prefers a bulky hydrophobic residue (Tyr). Based upon this and other information, peptide inhibitors have been designed that display both high potency and specificity. The tetrapeptide aldehyde Ac-Tyr-Val-Ala-Asp-CHO (which mimics the Tyr-Val-His-Asp caspase-1 recognition sequence within pro-IL-1β) is a potent inhibitor of caspase-1 (Ki=0.056 nM) but a poor inhibitor of caspase-3 (Ki=1960 nM). In contrast, the Ac-Asp-Glu-Val-Asp-CHO tetrapeptide aldehyde (which mimics the caspase-3 optimum recognition site) is a very potent inhibitor of caspase-3 (Ki=0.23 nM) but is a significantly weaker inhibitor of caspase-1 (Ki=18 nM) (Garcia-Calvo et. al. 1998, J. Biol. Chem. 273(49):32608-2613). Caspase inhibitors can be either reversible or irreversible, depending upon the nature of the “warhead” that attacks the active site cysteine. Peptide aldehydes, nitrites and ketones are potent reversible inhibitors, while compounds that form thiomethylketone adducts with the active site cysteine (e.g. peptide (acyloxy)methylketones) are potent irreversible inhibitors.
Excessive apoptosis is associated with a wide range of human diseases, and the importance of caspases in the progression of many of these disorders has been demonstrated with both small molecule and peptide-based inhibitors as well as by genetic approaches. Caspase inhibitors have been suggested to offer therapeutic benefit in numerous acute disorders, such as cardiac and cerebral ischemia/reperfusion injury (e.g. stroke), spinal cord injury, traumatic brain injury, organ damage during transplantation, liver degeneration (as caused, for example, by hepatitis), sepsis, bacterial meningitis and a number of dermatological conditions. There are also a wide range of chronic disorders in which excessive apoptosis is implicated, such as neurodegenerative diseases (e.g. Alzheimer's disease, polyglutamine-repeat disorders such as Huntington's Disease, Down's Syndrome, spinal muscular atrophy, multiple sclerosis, Parkinson's disease), immunodeficiency diseases (e. g. HIV), arthritis, atherosclerosis, diabetes, alopecia, and aging. Caspase inhibitors could also be used to extend the lifespan of purified blood products to be used for transfusions, or to enhance the lifespan of donated organs before transplantation. Thus, small molecule inhibitors of either Group I, II, or III caspases are likely to have tremendous therapeutic benefit (McBride et al. 1999, Emerging Ther. Targets 3(3):391-411; Talanian et al. 2000, J. Med. Chem. 43(18):3351-3371).